Anti-CREB抗体[LB9] (ab178322)

Western blot: Anti-CREB1 antibody [LB9] (ab178322) staining at 1/500 dilution, shown in green; Rabbit anti-ACTN4 [EPR2533(2)] (ab108198) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab178322 was shown to bind specifically to CREB1. A band was observed at 40 kDa in wild-type A549 cell lysates with no signal observed at this size in CREB1 knockout cell line. To generate this image, wild-type and CREB1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: CREB knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab178322 observed at 44 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab178322 was shown to recognize CREB when CREB knockout samples were used, along with additional cross-reactive bands. Wild-type and CREB knockout samples were subjected to SDS-PAGE.  Ab178322 and ab18058 (loading control to Vinculin) were diluted at 1/250 and 1/10,000 dilution respectively and incubated overnight at 4C. Blots were developed with IRDye® 800CW Goat anti-Rabbit IgG (H + L) and IRDye® 680 Goat anti-Mouse IgG (H + L) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of deparaffinized Human tonsil tissue labeling CREB with ab178322 at 1/100 dilution followed by DAB staining.

Immunoprecipitation analysis of CREB was performed on untreated 293T cells. The antigen:antbody complex was formed by binding 500μg whole cell lysate with 2μg ab178322 and captured on 50μl Protein A/G Plus Agarose. 

Lane 1: 293T lysate

Lane 2: IP of 293T lysate

WB detection used ab178322 at 1/1000 followed with a goat anti-mouse-HRP secondary antibody at 1/20,000.

Immunohistochemical analysis of deparaffinized Human bladder cancer (left) and rectal cancer (right) tissues labeling CREB with ab178322 at 1/200 dilution followed by DAB staining.

Immunofluorescent analysis of untreated HeLa cells (formalin-fixed, 0.1% Triton X-100 permeabilized) labeling CREB with ab178322 at 1/400 dilution (green) followed with DyLight 488 goat anti-mouse IgG secondary antibody at 1/400 dilution. Nuclei (blue) were stained with Hoechst 33342 dye.

Immunofluorescent analysis of untreated HeLa cells (formalin-fixed, 0.1% Triton X-100 permeabilized) labeling CREB with ab178322 at 1/500 dilution (green) followed with DyLight 488 goat anti-mouse IgG secondary antibody at 1/400 dilution. Nuclei (blue) were stained with Hoechst 33342 dye.